To keep track of the amount of a cholesterol synthesized in the body is very important for diagnoses of various pathological conditions and the like. In the body, a cholesterol is synthesized by the mevalonate pathway from acetyl coenzyme A via 3-hydroxymethylglutaryl coenzyme A (hereinafter also referred to as HMG-CoA), mevalonic acid (hereinafter also referred to as MVA), and the like. As the conversion of HMG-CoA to MVA catalyzed by hydroxymethylglutaryl coenzyme A reductase is a rate-determining step in this mevalonate pathway, the amount of a cholesterol synthesized in the body can be estimated by measuring the amount of mevalonic acid (Non Patent Literature 1). So far, MVA in a biological sample has been measured by a radioenzyme assay (Non Patent Literature 2), gas chromatography-mass spectrometry (GC-MS) (Non Patent Literature 3), liquid chromatography-mass spectrometry (LC-MS) (Non Patent Literature 4), an assay using antibody (Non Patent Literature 5), and the like. As shown in the above-mentioned documents, however, serum MVA concentrations are very low (63 to 200 nM [Non Patent Literature 1], 20 to 75 nM [Non Patent Literature 2], 18 nM [Non Patent Literature 3], 7.7 to 86.2 nM [Non Patent Literature 6]), and measurement of serum MVA concentrations by a colorimetric assay using an enzyme has not been reported so far. As in the case of MVA, HMG-CoA is also thought to be important as an indicator for the cholesterol metabolism in the body, but measurement of HMG-CoA concentrations by a colorimetric assay using an enzyme has not been reported so far.
Mevalonic acid has two optical isomers, which are expressed as D-mevalonic acid and L-mevalonic acid by the D/L notation and as R-mevalonic acid and S-mevalonic acid by the R/S notation. While D-mevalonic acid (may be expressed as R-mevalonic acid) is metabolized and it can serve as a substrate of mevalonate kinase and hydroxymethylglutaryl coenzyme A reductase in the body, L-mevalonic acid (may be expressed as S-mevalonic acid) is not metabolized in the body.
In the present specification, a term “D,L-mevalonic acid” or “D,L-MVA” represents racemic mevalonic acid, which is a mixture of the D form and the L form. When a term “mevalonic acid” or “MVA” is simply used in the present specification, the term represents D-mevalonic acid or R-mevalonic acid.
Similarly, 3-hydroxymethylglutaryl coenzyme A also has two optical isomers, which are expressed as D-3-hydroxymethylglutaryl coenzyme A and L-3-hydroxymethylglutaryl coenzyme A by the D/L notation and as R-3-hydroxymethylglutaryl coenzyme A and S-3-hydroxymethylglutaryl coenzyme A by the R/S notation. While D-3-hydroxymethylglutaryl coenzyme A (may be expressed as S-3-hydroxymethylglutaryl coenzyme A) is metabolized (can serve as a substrate of hydroxymethylglutaryl coenzyme A reductase) in the body, L-3-hydroxymethylglutaryl coenzyme A (may be expressed as R-3-hydroxymethylglutaryl coenzyme A) is not metabolized in the body.
In the present specification, a term “D,L-3-hydroxymethylglutaryl coenzyme A” or “D,L-HMG-CoA” represents racemic 3-hydroxymethylglutaryl coenzyme A, which is a mixture of the D form and the L form. When a term “3-hydroxymethylglutaryl coenzyme A” or “HMG-CoA” is simply used, the term represents D-3-hydroxymethylglutaryl coenzyme A or S-3-hydroxymethylglutaryl coenzyme A.
Furthermore, coenzyme A (hereinafter also referred to as CoA) is thought to be important as an indicator or the like of lipid metabolism in the body. However, convenient measurement of CoA concentrations by a colorimetric assay using an enzyme has not been reported so far.
An enzyme cycling Method has been reported as a method for measuring the concentration of an analyte with high sensitivity by a colorimetric assay using an enzyme. The enzyme cycling method is a method of amplifying a signal derived from an analyte A by an enzyme cycling reaction involving a hydrogen acceptor X and a hydrogen donor Y (here, the hydrogen donor Y and the reduced hydrogen acceptor X are not the same substance). The outline of the enzyme cycling method is represented by a combination of the following Reaction Formula 3:
and Reaction Formula 4:
Here, A is an analyte, the hydrogen donor Y and the reduced hydrogen acceptor X are not the same substance, and an enzyme that catalyzes Reaction Formulas 3 and 4, a hydrogen acceptor X, and a hydrogen donor Y are added to a test solution containing the analyte A to bring about the above-mentioned enzymatic reactions. The analyte A is cycled between A and A (oxide) during the reaction, and the reduced hydrogen acceptor X and the oxidized hydrogen donor Y are produced depending on the number of cycles. A signal derived from the analyte A is therefore amplified, and the analyte A can be measured with high sensitivity by colorimetrically measuring the amount of the reduced hydrogen acceptor X, the oxidized hydrogen donor Y, the decreased hydrogen acceptor X, or the decreased hydrogen donor Y.
In general, when the amount of an enzyme added to a reaction mixture is increased in an enzyme cycling reaction, the number of enzyme cycling reactions per unit time is increased, thereby improving sensitivity. However, it is impossible to add an enzyme to a reaction mixture in a certain amount or more, or to improve measurement sensitivity due to: 1) the reaction rate constant (kcat) of an enzyme involved in a reaction; 2) the amount of an enzyme that can be dissolved in a reaction mixture; 3) purity of the enzyme used; and the like. Therefore, the enzyme cycling reaction has a lower limit of the measurable concentration of an analyte. When the hydrogen acceptor X is oxidized thio-nicotinamide-adenine-dinucleotide (hereinafter also referred to as T-NAD) or oxidized thio-nicotinamide-adenine-dinucleotide phosphate (hereinafter also referred to as T-NADP), and the hydrogen donor Y is reduced nicotinamide adenine dinucleotide (hereinafter also referred to as NADH) or reduced nicotinamide adenine dinucleotide phosphate (hereinafter also referred to as NADPH) in the above-mentioned enzyme cycling method, the lower limit of the measurable concentration of an analyte is usually approximately 1 to 10 μM (Patent Literatures 1 and 2 and Non Patent Literatures 7 and 8).
Examples of the lower limit of the measurable concentration in a highly sensitive assay using the enzyme cycling method include: 0.2 μM when the analyte A is cholic acid, and the enzyme that catalyzes the enzyme cycling reaction is 3α-steroid dehydrogenase (Patent Literature 3); 0.2 μM when the analyte A is glucose-6-phosphate, and the enzyme that catalyzes the enzyme cycling reaction is glucose-6-phosphate dehydrogenase (Patent Literature 4); and 0.1 μM when the analyte A is a cholesterol and the enzyme that catalyzes the enzyme cycling reaction is cholesterol dehydrogenase (Non Patent Literature 9). Further, when the hydrogen acceptor X is oxygen and the hydrogen donor Y is NADH or reduced NADPH, concentrations to the lower limit of 0.03 μM could be measured by using glycerol-3-phosphate as an analyte, which is obtained by degrading lysophosphatidic acid with lysophosphatidic acid lipase, and using glycerol-3-phosphate oxidase and glycerol-3-phosphate dehydrogenase as enzymes catalyzing a cycling reaction and detecting hydrogen peroxide, which is a reduced hydrogen acceptor X, with peroxidase, 4-aminoantipyrine, and TOOS (Non Patent Literature 10).
In particular, known enzyme cycling reactions in which the hydrogen acceptor X is T-NAD or T-NADP and the hydrogen donor Y is NADH or NADPH are, for example: a reaction represented by a combination of the following Reaction Formula 5:
and the following Reaction Formula 6:
using a dehydrogenase for the analyte A (Patent Literatures 1, 3, and 4 and Non Patent Literatures 7 and 8); a reaction represented by a combination of the following Reaction Formula 7:
and the following Reaction Formula 8:
wherein the analyte is glutamic acid, α-ketoglutaric acid, or ammonia, and a glutamate dehydrogenase is used for these analytes (Patent Literature 2); or a reaction represented by a combination of the following Reaction Formula 9:
and the following Reaction Formula 10:
wherein the analyte is D-glyceroaldehyde-3-phosphate, inorganic phosphorus, or 1,3-diphosphoglyceric acid, and a D-glyceroaldehyde-3-phosphate dehydrogenase is used for these analytes (Patent Literature 5), and the like. Furthermore, it has been reported in Patent Literature 2 that the lower limit of the ammonium chloride that can be measured is 40 μM, the lower limit of the L-glutamic acid that can be measured is 40 μM, and the lower limit of L-leucine that can be measured is 4 μM in an enzyme cycling reaction in which leucine dehydrogenase was used instead of glutamate dehydrogenase, and glutamic acid was replaced with leucine and α-ketoglutaric acid was replaced with 2-oxoisocaproate in Reaction Formulas 7 and 8. It has been reported in Patent Literature 4 that the lower limit of the phosphoric acid that can be measured is 10 μM, and that the lower limit was 0.2 μM when measurement was performed using 3-phosphoglycerate kinase after 3-phosphoglyceric acid was converted into 1,3-diphosphoglyceric acid.